Over the past years, unscheduled mitosis has been considered to be a major characteristic of either benign or malignant neoplastic disease. Cancer development is believed to be due to a loss of control in the cellular strategies that regulate the cell cycle.
The mammalian cell cycle is believed to be driven by the sequential activation and deactivation of various cyclin/cyclin-dependent kinase (cdk) pairs (MacLachlan et al. (1995) Crit. Rev. in Eukary. Gene Ex. 5:127-56). The prototypic cell cycle kinase, cdc2, was originally discovered in yeast and found to be involved in regulating both DNA replication and mitosis. Higher organisms, however, were found to possess a more complex system monitoring these events.
One of several kinases involved, cyclin dependent kinase 2 (cdk2), is necessary for mammalian cells to pass the restriction point and onto DNA replication (Elledge, S. J. and Spottswood, M. R. (1991) EMBO J. 10:2653-2659). Activation and deactivation of this kinase is critical to the precise timing of the onset of S phase, after which a cell is committed to divide. Temporal association with cyclins A or E depict when the kinase will be active, and therefore phosphorylates the substrates involved in DNA replication. Over the last few years, several mechanisms besides cyclin binding have been discovered that control cdk activity. In addition to differences in phosphorylation status and cofactor binding, a growing number of low molecular weight proteins are now known to bind to and inhibit cdk kinase activity (Xiong, Y. (1996) Biochim. Biophys. Act. 1288:1-5; Sherr, C. J. and Roberts, J. M. (1995) Genes Dev. 9:1149-63). The CIP/KIP family (p21, p27, p57) and the INK family (p15, p16, p18, p19) of cdk inhibitors bind either the cyclin or directly to the cdk and suppress its kinase activities until cell cycle progression may continue. The INK family have a specificity for the early G1 kinases cdk4 and cdk6 complexed with D-type cyclins, while the CIP family is partial to cdk2 bound to either cyclin A or E. When overexpressed in a variety of cell types, these inhibitors are able to halt growth and arrest the cells in G1 phase. Accordingly, these are temporally regulated proteins, with a preference for potently inhibiting G1 cyclin-cdk pairs. The loss of several of these proteins has been found to be a critical step in oncogenesis. For example, the p16 and p15 genes both lie in a region found deleted in some 75% of all melanoma cases (Kamb et al. (1994) Science 264:436-440; Nobori et al. (1994) Nature 368:753-756). It appears that cdk inhibition is essential for normal cell cycle progression.
The retinoblastoma family consists of a group of genes, the proteins of which are also involved in eukaryotic cell cycle homeostasis. Proteins encoded by these genes are often referred to as "pocket proteins" due to their unique tridimensional structure. This structure is responsible for most of the specific and functionally relevant protein-protein interactions in which these molecules are involved. At present, the family consists of three members including the retinoblastoma (RB) gene which codifies a protein called pRb, p107 which codifies a protein called p107, and p130, also referred to as pRb2/p130, which codifies a protein called pRb2/p130.
All three pocket proteins are localized mainly in he nuclear compartment of the cells (Lee et al. (1987) Nature 329:642-645; Ewen et al. (1991) Cell 66:1155-1164; Baldi et al. (1995) J. Cell. Biochem. 59:402-408). Each protein structure consists basically of an N-terminal portion, the pocket structure subdivided into domain A, spacer and domain B, and a C-terminal portions, also called domain C. The pocket functional domains A and B are the most conserved and have been taught to responsible for most of the interactions involving either endogenous proteins or viral oncoproteins (Paggi et al. J. Cell. Biochem. (1996) 62:418-430).
The Rb protein is most well known for its interaction with and inhibition of the E2F transcription factor. It is believed that E2F is able to transactivate several genes whose products are necessary for DNA replication to occur. However, when bound to pRb, this transactivating ability is lost (Chellappan et al. (1991) Cell 65:1053-61). pRb is also found lost in several types of cancers, including retinoblastoma. The Rb related proteins p107 and pRb2/p130 have been found to have similar, yet distinctive qualities from pRb (Ewen et al. (1991) Cell 66:1155-64; Mayol et al. (1993) Oncogene 8:2561-6; Li et al. (1993) Genes Dev. 7:2366-77; and Hannon et al. (1993) Genes Dev. 7:2378-91). While pRb, p107, or pRb2/p130 overexpression can drive cancer cells to growth inhibition or arrest (Huang et al. (1988) Science 242:1563-1566; Bookstein et al. (1990) Science 247:712-715; Antelman et al. (1995) Oncogene 10:697-704; and Claudio et al. (1994) Cancer Res. 54:5556-5560), certain tumor cells are not equally responsive to any pocket protein. For example, the SAOS-2 human osteosarcoma cell line, which possesses a truncated non-functional pRb molecule is definitely sensitive to pRb and p107 growth suppressive properties. On the other hand, while pRb2/p130 overexpression slows down SAOS-2 growth, this protein has also been able to inhibit proliferation in the T98G human glioblastoma multiform and in the MCF-7 human mammary adenocarcinoma cell lines, while neither pRb nor p107 showed any inhibitory effect. Both T98G and MCF-7 cell lines display homozygous deletion of the p16 .sup.INK4A gene, a cdk4 and cdk6 inhibitor. Accordingly, is has been suggested that pocket protein Rb and p107 are become extensively phosphorylated by specific cyclin/cdk complexes, thus becoming functionally inactive in blocking the cell cycle. It is believed, however, that pRb2/p130 which has been found to be coupled to cdk2 is able to overcome p16.sup.INK4A homozygous deletion, bringing about effective growth inhibition in these cell lines (Paggi et al. J. Cell. Biochem. (1996) 62:418-430).
The functional domain of the Rb and p107 proteins have been mapped through both genetic and biochemical means (Hu et al. (1990) EMBO J. 9:1147-1155; Ewen et al. (1991) Cell 66:1155-1164; Ewen et al. (1992) Science 255:85-87). An approximately 400 amino acid fragment of Rb and p107, termed the Rb pocket, is responsible for association of these proteins with the DNA tumor virus oncoproteins and cellular ligands. Within this domain are six regions of extensive sequence similarity. Similarly, the cyclins share a large region of sequence similarity spanning approximately 87 amino acids which has been designated that the "cyclin box" (Pines et al. (1989) Cell 58:833-846). This domain is believed to be important in Rb and p107 protein:protein interactions. In fact peptides having sequences comprising a portion of this domain have been proposed as inhibitors of cell growth and proliferation (U.S. Pat. No. 5,625,031).
It has now been found that the inhibitory activity of the related Rb protein, pRb2/p130, occurs not through this cyclin box, but rather through a different and separate domain of the spacer region. Further, increased expression of pRb2/p130 during various cellular processes was demonstrated to be associated with decreased kinase activity of cdk2. For example, pRb2/p130 protein levels are increased upon differentiation of myocytes, coincident with cdk2 kinase inhibition. This is quite different from the related p107 protein, levels of which have been shown to decrease. Accordingly, it is believed that pRb2/p130 acts not only to bind and modify E2F activity, but also to inhibit cdk2 kinase activity in concert with p21 in a manner different from other Rb proteins.